Title of Project: Defining mechanisms for lipid transport across capillary endothelial cells Our objective is to understand how lipids from triglyceride-rich lipoproteins (TRLs) move across capillary endothelial cells towards parenchymal cells. Our interest in this topic-the least understood area within plasma triglyceride metabolism-arose from our efforts to understand molecular mechanisms for the intravascular processing of TRLs. During the past few years, we showed that GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is solely responsible for shuttling lipoprotein lipase (LPL) from the interstitial spaces to its site of action in the capillary lumen. More recenty, we showed that the LPL-GPIHBP1 complex is critical for the margination of TRLs along capillaries (so that the LPL-mediated processing of TRLs can proceed). The movement of lipid nutrients across capillaries to parenchymal cells is crucial for delivering fuel to vital organs an lipids for storage in adipose tissue. Unfortunately, there are few insights into this process. No one understands: (1) whether the fatty acid products of lipolysis simply diffuse across endothelial cells; (2) whether lipids move across endothelial in the very same vesicles that shuttle GPIHBP1 and LPL; (3) whether intact TRLs move across endothelial cells to the subendothelial spaces; and (4) whether binding of fatty acids by CD36 on endothelial cells is required for lipid transport across capillaries. Also, no one understands how lipids move across capillaries in other vertebrates (e.g., birds, fish). In those organisms, GPIHBP1 is absent and LPL appears to be located largely, if not exclusively, in the extravascular spaces (i.e., it is not associated with capillaries). In other vertebrates, we suspect that the TRLs might be transported across capillaries to the subendothelial spaces-to where the LPL is located. An improved understanding of TRL metabolism in other vertebrates will likely yield insights into accessory mechanisms for TRL processing in mammals. One of the main reasons for the slow progress in understanding lipid transport across capillaries is that there has been no way to visualize lipid transport. Seeing how lipids move across capillaries is crucial for deciphering molecular mechanisms and designing testable hypotheses. Fortunately, we have overcome the imaging roadblock. During the past two years, we have used NanoSIMS and backscattered electron (BSE) imaging to create high-resolution images of TRLs as they marginate along capillaries and as the TRL lipids move across endothelial cells to parenchymal cells. These studies have demonstrated that some TRL lipids move across endothelial cells in vesicles, but additional high-resolution imaging studies are required to determine if diffusion of fatty acids along plasma membranes-or transport of lipids across the cytosol-is involved. The same methods can be used to define the role of specific proteins (e.g., CD36) in lipid transport across endothelial cels. For the next five years, we will use NanoSIMS and BSE imaging to define the cellular and molecular mechanisms for lipid transport across capillaries. We will also define the in vivo functional relevance of CD36 for the transport of TRL-derived lipids across capillary endothelial cells.